This invention relates to a reliable, precise, and highly sensitive analytical system for detecting biochemical agents that catalyze a redox potential change.
By way of background, a light-addressable potentiometric sensor ("LAPS")and a rapid capture immunoassay system have been developed by the Molecular Devices Corporation of Sunnyvale, Calif. The rapid capture immunoassay system, which is commercially available under the Threshold.RTM. trademark, uses LAPS technology to perform a multiplicity of pH-based immunoassays in small volumes with high sensitivity. In addition to the detection and quantitation of hydrogen ions, specific ion-sensing membranes may be deposited to fabricate sensing sites to detect and quantitate several other basic analytes including, for example, aqueous ions such as Na.sup.+, K.sup.+, Ca.sup.++, and Cl-. Deposition of a valinomycin-containing polyvinyl-chloride membrane results in a spatially-resolved, K.sup.+ ion sensor. Various gases, including hydrogen, ammonia, hydrogen sulfide, ethylene and ethanol, can also be detected and quantitated. Multiple assays of a single basic analyte, or of several basic analytes, may be monitored simultaneously at a multiplicity of sites on a single dielectric-coated semiconductor surface of the LAPS device.
In the small volume detection chamber of the Threshold.RTM. immunoassay detection system, the lower limit of detection of pH-altering enzymes is determined by the surface pH-buffer capacity, pH-buffer concentration, and the volume of the reaction chamber. A nitrocellulose membrane is utilized to specifically capture analyte molecules which are then labeled with an enzyme capable of catalyzing a pH-changing reaction. One limitation to the sensitivity for detection of such enzymes is the fact that the nitrocellulose membrane itself has a fixed site buffer capacity equivalent to 2.3 mM buffer concentration. Impurities retained on the membrane from samples tend to further increase the buffer capacity. A sensitivity limitation of the Threshold.RTM. Immunoassay System is that proteinaceous materials adsorbed onto the membrane from samples tend to have many proton-dissociable sites. Therefore, it is difficult to reliably improve sensitivity by reducing the surface pH-buffer capacity of the membrane.
The LAPS device can also be configured to monitor redox potential. Multiple, spatially separate, redox potential measurements can be made by depositing thin pads of metallic gold (or other noble metal) on the dielectric. When the electrolyte solution contains a redox pair such as ferricyanide-ferrocyanide, the potential of the nobel metal is determined by the standard redox potential of the redox pair and the ratio of activities of the members of the redox pair in accordance with the Nernst equation. Intensity-modulated illumination of a region of semiconductor adjacent the nobel metal produces an alternating photocurrent similar to that observed with the pH-sensing device. In this case, however, the relationship between the measured alternating photocurrent, I, and the applied bias potential, .PSI., responds to changes in redox potential of the electrolyte.
The Threshold.RTM. immunoassay detection system has been modified to quantitate enzymes that generate redox-active products. The modified Threshold.RTM. systems use the LAPS device, modified to monitor redox potential, as well as selected pairs of enzyme substrate and soluble redox mediators to provide sensitive detection and quantitation of enzymes such as alkaline phosphatase (ALP) and horseradish peroxidase (HRP). The enzyme substrate is chosen to react rapidly in the presence of the enzyme and to generate a redox active product. The soluble redox mediator functions to react with the redox-active product and carry electrons to or from a metal electrode or the metallic gold pads on the LAPS device.
The Threshold.RTM. immunoassay detection system modified to detect redox potential may have a higher sensitivity for detection of small quantities of enzyme provided that the redox buffer capacity on the nitrocellulose membrane is lower than the pH-buffering capacity. Sensitivity could also be improved by decreasing the volume of the reaction chamber. Decreasing the volume of the reaction chamber to a very small volume, however, makes it difficult to precisely maintain a constant volume during repetitive measurements. Because the measured rate of change in potential is inversely proportional to volume and directly proportional to the amount of enzyme present, precise determination of the quantity of enzyme requires precise control of volume. Such control, however, become increasingly difficult as the microvolume becomes vanishingly small.
One way to overcome this limitation is to make the enzyme quantitation independent of volume by providing a feedback current by means of a feedback electrode to keep the redox potential constant. In this way, the amount of feedback current required to maintain the potential constant may be measured and, in turn, used to determine the amount of enzyme or enzymatic activity present. Alternatively, the redox potential need not be kept constant, but instead may be allowed to reach a new steady-state. Thus, the current, or charge, conducted by a feedback electrode to maintain a new steady-state potential in the presence of an enzymatic reaction may be used to quantitate the amount of eneymatic activity present.